This invention relates to production of a modified hemoglobin composition, used as a blood substitute and a blood plasma expander. There is a critical need within the medical industry for blood substitutes and blood volume expanders. This need occurs not only because of the shortage of donor blood in blood banks, but also because of many problems that commonly exist with donor bloodbank practices. For example, there is an increasingly significant risk of disease transmission such as acquired immunodeficiency syndrome, commonly referred to as "AIDS", and even much more commonly, a real hepatitis risk. The shelf life of whole blood is also relatively short, not usually lasting longer than 30 days. There is also the problem of the need for blood typing, etc. with donated whole blood samples.
Accordingly, there is a very real and continuing need which has existed for some time, for blood substitutes or blood plasma expanders which can be conveniently prepared from a base hemoglobin source, such as outdated blood. This invention has as its primary objective, the fulfillment of this continuing need.
Currently there are two available possible routes for blood substitutes and blood plasma expanders which are being investigated. The first is fluorocarbons and the second is modified hemoglobins. The modified polyhemoglobins are represented by U.S. Pat. No. 4,001,401. Fluorocarbons are also receiving much active investigation at the present. However, it is believed unlikely that fluorocarbons will every successfully take over the market for blood substitutes or blood plasma expanders because these are known to at times block the natural immune system. In addition, the use of fluorocarbons is limited to situations in which high partial pressures of oxygen can be administered. They do not have a sufficiently high oxygen binding capacity for use under normal environmental conditions. Thus, while currently available materials do represent a contribution and some advancement in medical sciences directed towards the concept of a blood substitute and blood plasma expander, there is currently nothing of significant commercial affect available on the market.
There is also the problem of not only developing an effective oxygen carrying blood substitute which will effectively release the oxygen for body use, but also developing a composition which will not be renally eliminated. A natural mammalian hemoglobin is a tetramer, which in plasma will in the oxy form have a tendency to split into two alpha-beta dimers, each having a molecular weight of approximately 32,000. These dimers are small enough to be filtered by the kidneys and be excreted, with the result being a potential for renal injury and a substantially decreased intravascular retention time.
It therefore becomes readily apparent that there is a continuing need for a therapeutic product useful as a blood substitute and blood plasma expander, which will effectively bind oxygen, but not bind it so tightly that it will not be released for body use; and, for development of a product which will not split into alpha-beta dimers, capable of rapid elimination by the renal route as well as loss from the circulation through capillary beds in other tissues. The composition of my prior application accomplishes the primary objective of fulfilling this expressed need.
Accordingly, another primary object of my prior invention was to prepare an effective blood substitute and blood plasma expander from modified hemoglobin.
Another objective of the prior invention was to prepare a blood substitute and blood plasma expander based on a derivative of hemoglobin cross-linked specifically between the alpha chains, at Lys 99 Alpha.sub.1 to Lys 99 Alpha.sub.2.
The composition of my prior invention was initially prepared as only a minor reaction product at yields of approximately 10% to 15% of theoretical. Such yields are too low to be of commercial significance, and must therefore be increased significantly if the composition of my prior invention is to be available at practical cost for use as a blood substitute and blood plasma expander.
A primary objective of the present invention is the production of the composition of my prior invention in high yields. In particular, at yields which are commercially significant, that is within the range of at least 50% to 60% of theoretical such that the alpha-alpha cross-linked hemoglobin composition can be prepared in yields that make it practical as a blood substitute and blood plasma expander.
A further objective of the present invention is to prepare the blood substitute and plasma expanders of my prior invention in the presence of an added polyanion such as 2,3-diphosphoglycerate, inositol hexaphosphate or inositol hexasulfate, it having been discovered that when the cross-linking reaction with deoxygenated hemoglobin described in my prior invention occurs in the presence of one of these polyanions, the yield of product is significantly increased from 10% to 15% up to as high as 60-70%. The formation of unwanted side products is correspondingly reduced. These results greatly simplify the purification of the cross-linked hemoglobin and make its production feasible on a commercial scale.